Summary: Vaccines are among the most cost-effective public health measures available today. Because many vaccines are administered to all healthy children, and development of herd immunity is an important contributor to their effectiveness, it is important for the public to have confidence that vaccines are safe. It is thus very important from a public health perspective to ensure that administered vaccines are free of known adventitious agents. The goal of this project is to develop new tests that may be reliably used to test vaccines for potential adventitious agents. Most current vaccine testing relies on highly sensitive tissue culture assays. Newer molecular techniques have not in general been adapted for this kind of testing, in part because of the high reliability and sensitivity of the tissue culture assays. However, these molecular techniques have the potential to detect agents that theoretically could yield negative tissue culture results. Thus, new molecular techniques are being used to develop assays that could detect potential viral adventitious agents. Polymerase chain reactions were used to screen vaccine lots for the presence of simian cytomegalovirus (SCMV) DNA. SCMV DNA was found in some lots of vaccine. However, viable SCMV could not be cultured from any vaccine lot. Improved tissue culture methods for detection of SCMV were investigated. A framework for using the results of laboratory studies to assess adventitious agent risk in a regulatory setting was developed. Future studies will focus on the development of non-specific assays, that detect adventitious viral nucleic acid based on specific features of contaminating viruses, without a clear need to know the DNA or RNA sequence o f the virus being detected. The current approach is to purify viral nucleic acids from biological samples and to use non-specific PCR methods to amplify those viral nucleic acids. These approaches are shown to be sensitive and very non-specific in nature. Continuing work will address improvement in sensitivity and application of these assays to relevant samples, as well as investigation of additional approaches to non-specific detection of viruses.